Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common inherited nephropathy, characterized by progressively enlarged cystic kidneys and leading to end stage renal disease. The ADPKD disease spectrum is associated with cardiovascular abnormalities and cysts in other organs, particularly the liver. ADPKD is caused by mutations in two genes, PKD1 and PKD2. In our previous studies in ADPKD, we have developed molecular tools for the mutation analysis of the PKD1 and PKD2 genes, described genotype- phenotype correlations in ADPKD cohorts (association of mutation position with severity of renal disease and with development of a vascular phenotype), and developed a scoring matrix to classify missense mutations in these two genes. Sequencing by Sanger method of the PKD1 and PKD2 genes uses a large number of amplicons, and it is focused around the coding exons and close intronic regions. Such approach does not explore deep introns and may be prone to allele drop-out due to the high number of primers used. We now propose to utilize the Illumina Genome Analyzer II next-generation sequencing platform as a tool for comprehensive mutation analysis of the PKD genes (Specific Aim 1). We propose to develop long-range PCR amplicons that cover the whole genomic structure of both the PKD1 and PKD2 genes. We will utilize these long-range PCR amplicons and next-generation sequencing for exploring all the deep intronic regions of the PKD1 and PKD2 genes in a group of 30 ADPKD patients, who are mutation-negative after sequencing by Sanger method. This will allow determining whether this group of mutation-negative ADPKD patients, likely enriched for atypical mutations, does carry deep intronic mutations affecting splicing, or whether allele drop-out occurred in these patients during Sanger analysis. We propose to evaluate all the intronic changes found in Specific Aim 1 by in silico and in vitro approaches (Specific Aim 2). Deep intronic variants found in this cohort will be evaluated for pathogencity using population data (the NCBI dbSNP, the pattern of normal intronic variation, and segregation analysis), to filter out common polymorphisms from private variants;in silico predictive tools (4 predictive tools), to predict whether splicing is likely affected by these variants;and in vitro functional assays (cDNA and minigene analysis), to verify if splicing is actually affected by the intronic variants selected through the two previous steps. Further characterization of this mutation-negative cohort will allow to investigate the role of deep intronic mutations in ADPKD, investigate new mechanisms by which splicing is regulated in the PKD genes, and analyze genotype-phenotype correlations. PUBLIC HEALTH RELEVANCE: Autosomal Dominant Polycystic Kidney Disease is a frequent inherited nephropathy that leads to end stage renal disease by mid-life. The disease is caused by mutations at two genes, PKD1 and PKD2. ADPKD type1 patients reach ESRD typically 20 years earlier than ADPKD type2 patients (54 years of age versus 74 years of age). Several hundreds different mutations account for ADPKD in both genes, and they are mostly specific to a single family. The PKD1 and PKD2 genes are large and complex genes. Complete analysis of these genes requires the amplification by PCR and sequencing by Sanger methods of a large number of fragments. However, such analysis is focused around coding exons and flanking introns, leaving deep introns unexplored. Allele drop-out may also occur when using a large number of PCR primers, due to the presence of SNPs in the target sequence. Sequencing by Sanger methods of large cohorts reveals that ~10% of ADPKD patients remain mutation-negative. These patients are likely to be enriched for atypical mutations, like deep intronic mutations that lead to abnormal splicing. Since deep introns are not currently explored, such mutation type has not yet been descried in ADPKD. Here we propose to utilize the Illumina Genome Analyzer II next-generation platform for deep sequencing a group of 30 ADPKD patients who are mutation-negative after extensive molecular characterization. We will utilize long-range amplicons to cover the whole genomic structure of both the PKD1 and PKD2 genes, and next-generation sequencing to obtain a complete molecular signature of both genes in these mutation-negative ADPKD patients. The intronic variants found in these patients will be evaluated using population data (the NCBI dbSNP and the pattern of normal intronic variation), in silico tools (to predict the activation of cryptic splicing sites) and in vitro assays (to functionally validate sequence variants strongly predicted to affect normal splicing). In conclusion, we propose to utilize deep genomic sequencing and multi-step validation of intronic variants in order to determine whether these mutation-negative ADPKD patients do carry a deep intronic variant. Such finding will validate this assay as a comprehensive genotyping assay for ADPKD, particularly in mutation- negative patients, and allow novel interesting genotype-phenotype correlations. The finding of deep intronic mutations affecting splicing may lead to new understanding on how proper gene splicing is regulated.